Pichia pastoris is a very widely used well-established industrial host for producing therapeutic recombinant proteins. Production of therapeutic recombinant proteins poses several problems such as clipping at N-terminal (Prabha et. al. 2009) or C-terminal, introduction of desired glycoforms (Wouter Vervecken et. al. 2004). There is a need for enhancing the protein productivity and quality to meet the growing demands of the market and the growing regulatory issues on biosimilarities. This can be achieved by co-expressing the molecular chaperones (Wei Zhang et. al. 2006), Co-expression of proteases to get the desired end product. In all these cases there is need for additional promoters for carrying out genetic manipulation to improve quality and quantity of the recombinant protein.
U.S. Pat. No. 6,033,898 discloses a DNA segment isolated from a Saccharomyces cerevisiae sorbitol dehydrogenase gene which is utilized to increase production of a heterologous polypeptide. U.S. Pat. No. 5,139,936 discloses a cloning vector containing a foreign gene and the yeast galactosidase (GAL1) regulatory region and promoter in position to increase expression of the foreign gene. U.S. Pat. No. 5,089,398 discloses the use of the promoter region from the glyceraldehyde-3-phosphate dehydrogenase to control expression of a foreign polypeptide in yeast.
Although several promoters, either constitutive or inducible are available for the Pichia expression system, AOX1 is employed in most studies and application. AOX1 promoter is a strong inducible promoter and has tight regulation by carbon sources, expression is highly repressed when Pichia pastoris is grown in the presence most of other carbon sources other than Methanol. Methanol is an inducer of AOX1 promoter, methanol is a hazardous substance due to its high flammability and toxicity, also the cells growing on methanol have a very high oxygen consumption which usually requires addition of pure oxygen to the culture, thus increasing the cost of the process and limiting the cultivation capacity at large scale (Monika Bollok et. al., Recent Patents on biotechnology, 3(2009) 192-201.)
Also host cellular protein (HCP) release from P. pastoris grown on methanol is high which is mainly derived during cell lysis but it occurs to a much lower extent upon growth with glucose as a carbon source. As a result cells maintain higher viability and higher purity of the secreted protein.
Further, the sorbitol dehydrogenase promoter identified in S. cerevisiae is 750 bps. Due to large base pair size, it is not so easy to carry out genetic manipulation as it leads to increase of the vector size. The instant disclosure aims at overcoming the issues mentioned above. Also, there is a need in the art to identify more promoters for the expression of heterologous recombinant proteins in Pichia pastoris which does not require any specific induction for expression of protein.